Project Summary/Abstract The molecular mechanisms by which embryos pattern the early neuroectoderm along the anterior- posterior (AP) body axis and the mechanisms by which Wnt signaling networks govern multiple aspects of development and adult homeostasis are fundamental biological questions. However, much remains to be discovered about each of these questions, especially during the early development of deuterostome embryos where Wnt signaling is essential for anterior-posterior neuroectoderm patterning. We have recently discovered that 3 different, but interconnected, Wnt signaling pathways form a Wnt network that is essential for patterning the neuroectoderm along the anterior-posterior axis in early sea urchin embryos. Comparison of functional and expression studies among multiple deuterostome species (i.e., echinoderms, hemichordates, chordates), including vertebrates, strongly suggests that aspects of this Wnt network are conserved. The long-term goal of the studies in our lab is to systematically characterize the extracellular, intracellular and transcriptional players involved in the Wnt network governing early AP neuroectoderm patterning in the sea urchin. The objective of this R15 is to establish the transcriptional gene regulatory network (GRN) activated downstream of the Wnt network and perform initial functional studies to uncover how extracellular and intracellular Wnt modulators influence the activity of the network. The central hypothesis is that there are key interactions among the signaling pathways at the extracellular, intracellular, and transcriptional level. The rationale is that by first generating the transcriptional GRNs we can better understand how the extracellular and intracellular Wnt network are integrated in the AP neuroectoderm patterning GRN. Aim1 will uncover the transcription factors activated by the Wnt/JNK and Wnt/PKC signaling pathways during AP neuroectoderm patterning using information from differential screens comparing wild type embryos with Wnt/JNK and Wnt/PKC knockdown embryos. Functional gene perturbation studies will be performed to establish the transcriptional GRN scaffold and to define key interactions between the Wnt pathways at the transcriptional level. Aim 2 will use gene perturbation analyses on putative extracellular and intracellular Wnt modulators in order to better characterize the pathway members used in the Wnt/JNK and Wnt/PKC transduction pathways, to identify possible interactions among these modulators between the pathways, and to learn how these modulators affect the emerging GRN created in Aim1. The proposed research is significant because it will be one of the few systematic studies conducted to determine how these Wnt networks influence development in an in vivo model system. It will also provide the baseline for comparative functional studies of the GRN in other deuterostome model species, including vertebrates, thereby filling in large gaps in our knowledge of the evolution of early AP patterning mechanisms.